L-type Ca2+ channels regulate various functions spanning from neuronal excitability to gene transcription. The long term interest of this grant is to determine the molecular mechanisms that control activity and functional availability of the L-type channel Cav1.2. Our strong preliminary data indicate that a-actinin binds directly to the central pore-forming Cav1.2 subunit. Calmodulin binds to the same region as a-actinin and displaces it from Cav1.2 in the presence but not absence of Ca2+. Knock-down of a-actinin with siRNA or expression of two different dominant negative a-actinin fragments lead to a -50% reduction in the current density of Cav1.2 and substantially reduce peripheral Cav1.2 localization as detected by immunofluorescence. We hypothesize that a-actinin fosters surface expression of Cav1.2 by inhibiting its endocytosis. We further hypothesize that displacement of a-actinin by Ca2+/calmodulin promotes endocytosis of Cav1.2. We will define residues on Cav1.2 that are critical for a-actinin but not CaM binding and vice versa. We will inhibit the interactions between a-actinin and Cav1.2 by: a) expressing dominant negative a-actinin constructs; b) knocking down a-actinin with siRNA (we already established the siRNAs); c) expressing Cav1.2 with residues mutated that are crucial for a-actinin binding. Whether surface expression is reduced following these manipulations as compared to control conditions will be evaluated by biochemical and cell biological methods. We will investigate the role of calmodulin in dislocating a-actinin from Cav1.2 upon Ca2+ influx, thereby perhaps promoting Cav1.2 internalization, by: a) overexpressing Ca2+ binding-deficient calmodulin mutants that act as dominant negative constructs for a number of ion channels with respect to the regulation of their gating by Ca2+/calmodulin, including Cav1.2; b) expressing mutant Cav1.2 that does not bind calmodulin but still interacts with a-actinin. Ca2+-dependent internalization will be compared between control conditions (e.g., overexpression of wt calmodulin or wt Cav1.2) and test conditions (e.g., dominant negative calmodulin or mutant Cav1.2). Cav1.2 channel activity is strongly increased in aged rats. The L-type channel inhibitor nimodipine impressively improves learning capabilities of aged rodents. Hence, an increase in Cav1.2 channel activity is thought to contribute to the etiology of senile symptoms and Alzheimer's disease. Cav1.2 has also been implicated in depression and anxiety disorders. Our work on the regulation of surface expression of Cav1.2 by the interplay between a-actinin and calmodulin will fill a critical gap in our understanding of how Cav1.2 surface expression and localization is regulated. It will thereby contribute to the development of treatment of these diseases. [unreadable] [unreadable] [unreadable]